Moslecular and biological characterization of human 4‐1BB and its ligands

Abstract

4-1BB was originally described as a cDNA expressed by activated murine T cells and subsequently demonstrated to encode a member of the tumor necrosis factor receptor family of integral membrane proteins. Recently, we identified and cloned a murine ligand for 4-1BB (mu4-1BB-L) and demonstrated it to be a member of an emerging family of ligands with structural homology to tumor necrosis factor. To characterize further the role of 4-1BB in the immune response we undertook to clone the human homologue of 4-1BB-L. However, attempts to isolate a cDNA encoding the human 4-1BB-L by cross-hybridization with the murine cDNA were unsuccessful. Therefore we first utilized cross-species hybridization to isolate a cDNA encoding human 4-1BB (hu4-1BB). A fusion protein consisting of the extracellular portion of hu4-1BB coupled to the Fc region of human immunoglobulin G1 (hu4-1BB.Fc) was then used to identify and clone a gene for human 4-1BB-L from an activated CD4+ T cell clone using a direct expression cloning strategy. Human 4-1BB-L shows 36% amino acid identity with its murine counterpart and maps to chromosome 19p13.3. Scatchard analysis demonstrated high-affinity binding of hu4-1BB.Fc to either native or recombinant human 4-1BB-L. Both monoclonal antibody to hu4-1BB and cells transfected with hu4-1BB-L induced a strong proliferative response in mitogen co-stimulated primary T cells. In contrast, ligation of 4-1BB on T cell clones enhanced activation-induced cell death when triggered by engagement of the TCR/CD3 complex.

Role of 4-1BB:4-1BB ligand in cancer immunotherapy

“…13 The molecular weight of the protein was calculated to be 27 kDa 15 and was shown to be 60% identical to murine 4-1BB (Fig 1c). 13 In the cytoplasmic domain, five regions of a.a. sequences were conserved between mice and human, indicating that these residues might be important for 4-1BB function. 13 …”

“…11 The human homologue of 4-1BB (CD137) was cloned from activated human T-cell leukemia virus type 1-transformed human T-lymphocytes library. 12 Human 4-1BB resides on chromosome 1p36 13 (Fig 1aii), in a cluster of related genes including TNFR2, CD30, OX40 and TAMP/Apo3, which have been shown to be mutated in several malignancies. 14 The human 4-1BB contains 255 a.a. with two potential N-linked glycosylation sites.…”

“…14 The human 4-1BB contains 255 a.a. with two potential N-linked glycosylation sites. 13 Hydrophobicity analysis predicted that a.a. 1-17 were a putative signal peptide, which was followed by an extracellular domain of 169 a.a. and then a transmem-brane region of 27 a.a. between positions 187-213, and finally a short intracellular domain of 42 a.a (Fig 1bii). 13 The molecular weight of the protein was calculated to be 27 kDa 15 and was shown to be 60% identical to murine 4-1BB (Fig 1c).…”

“…13 Hydrophobicity analysis predicted that a.a. 1-17 were a putative signal peptide, which was followed by an extracellular domain of 169 a.a. and then a transmem-brane region of 27 a.a. between positions 187-213, and finally a short intracellular domain of 42 a.a (Fig 1bii). 13 The molecular weight of the protein was calculated to be 27 kDa 15 and was shown to be 60% identical to murine 4-1BB (Fig 1c). 13 In the cytoplasmic domain, five regions of a.a. sequences were conserved between mice and human, indicating that these residues might be important for 4-1BB function.…”

“…13 In the cytoplasmic domain, five regions of a.a. sequences were conserved between mice and human, indicating that these residues might be important for 4-1BB function. 13 …”